Qinwen Mao

siRNA-mediated gene silencing in vitro and in vivo

RNA interference is now established as an important biological strategy for gene silencing, but its application to mammalian cells has been limited by nonspecific inhibitory effects of long dsRNA on translation. Here, we describe a viral-mediated delivery mechanism that results in specific silencing of targeted genes through expression of small interfering RNA (siRNA). We establish proof of principle by markedly diminishing expression of exogenous and endogenous genes in vitro and in vivo in brain and liver, and further apply this strategy to a model system of a major class of neurodegenerative disorders, the polyglutamine diseases, to show reduced polyglutamine aggregation in cells. This viral-mediated strategy should prove generally useful in reducing expression of target genes to model biological processes or to provide therapy for dominant human diseases.

RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia

The dominant polyglutamine expansion diseases, which include spinocerebellar ataxia type 1 (SCA1) and Huntington disease, are progressive, untreatable, neurodegenerative disorders. In inducible mouse models of SCA1 and Huntington disease, repression of mutant allele expression improves disease phenotypes. Thus, therapies designed to inhibit expression of the mutant gene would be beneficial. Here we evaluate the ability of RNA interference (RNAi) to inhibit polyglutamine-induced neurodegeneration caused by mutant ataxin-1 in a mouse model of SCA1. Upon intracerebellar injection, recombinant adeno-associated virus (AAV) vectors expressing short hairpin RNAs profoundly improved motor coordination, restored cerebellar morphology and resolved characteristic ataxin-1 inclusions in Purkinje cells of SCA1 mice. Our data demonstrate in vivo the potential use of RNAi as therapy for dominant neurodegenerative disease.

The HIV Tat protein transduction domain improves the biodistribution of β-glucuronidase expressed from recombinant viral vectors

Treatment of inherited genetic diseases of the brain remains an intractable problem. Methods to improve the distribution of enzymes that are injected or expressed from transduced cells will be required for many human brain therapies. Recent studies showed that a peptide, the protein transduction domain (PTD) from HIV Tat, could improve the distribution of cytoplasmic reporter proteins when administered systemically as fusion proteins or cross-linked chimeras. The utility of this motif for noncytoplasmic proteins has not been determined. Here, we tested how the Tat motif affected uptake and biodistribution of the lysosomal enzyme β-glucuronidase, the protein deficient in the disease mucopolysaccharidosis VII, when expressed from viral vectors. The Tat motif allowed for mannose-6-phosphate (M6P) independent uptake in vitro and significantly increased the distribution of β-glucuronidase secreted from transduced cells after intravenous or direct brain injection in mice of recombinant vectors. Thus, enzymes modified to contain protein transduction motifs may represent a general strategy for improving the distribution of secreted proteins following in vivo gene transfer.

Recombinant Human Adenovirus: Targeting to the Human Transferrin Receptor Improves Gene Transfer to Brain Microcapillary Endothelium

ABSTRACT Some inborn errors of metabolism due to deficiencies of soluble lysosomal enzymes cause global neurodegenerative disease. Representative examples include the infantile and late infantile forms of the ceroid lipofuscinoses (CLN1 or CLN2 deficiency, respectively) and mucopolysaccharidoses type VII (MPS VII), a deficiency of β-glucuronidase. Treatment of the central nervous system component of these disorders will require widespread protein or enzyme replacement, either through dissemination of the protein or through dissemination of a gene encoding it. We hypothesize that transduction of brain microcapillary endothelium (BME) with recombinant viral vectors, with secretion of enzyme product basolaterally, could allow for widespread enzyme dissemination. To achieve this, viruses should be modified to target the BME. This requires (i) identification of a BME-resident target receptor, (ii) identification of motifs targeted to that molecule, (iii) the construction of modified viruses to allow for binding to the target receptor, and (iv) demonstrated transduction of receptor-expressing cells. In proof of principal experiments, we chose the human transferrin receptor (hTfR), a molecule found at high density on human BME. A nonamer phage display library was panned for motifs which could bind hTfR. Forty-three clones were sequenced, most of which contained an AKxxK/R, KxKxPK/R, or KxK motif. Ten peptides representative of the three motifs were cloned into the HI loop of adenovirus type 5 fiber. All motifs tested retained their ability to trimerize and bind transferrin receptor, and seven allowed for recombinant adenovirus production. Importantly, the fiber-modified viruses facilitated increased gene transfer (2- to 34-fold) to hTfR expressing cell lines and human brain microcapillary endothelia expressing high levels of endogenous receptor. Our data indicate that adenoviruses can be modified in the HI loop for expanded tropism to the hTfR.

A Mouse Model of Classical Late-Infantile Neuronal Ceroid Lipofuscinosis Based on Targeted Disruption of the CLN2 Gene Results in a Loss of Tripeptidyl-Peptidase I Activity and Progressive Neurodegeneration

Mutations in the CLN2 gene, which encodes a lysosomal serine protease, tripeptidyl-peptidase I (TPP I), result in an autosomal recessive neurodegenerative disease of children, classical late-infantile neuronal ceroid lipofuscinosis (cLINCL). cLINCL is inevitably fatal, and there currently exists no cure or effective treatment. In this report, we provide the characterization of the first CLN2-targeted mouse model for cLINCL. CLN2-targeted mice were fertile and apparently healthy at birth despite an absence of detectable TPP I activity. At ∼7 weeks of age, neurological deficiencies became evident with the onset of a tremor that became progressively more severe and was eventually accompanied by ataxia. Lifespan of the affected mice was greatly reduced (median survival, 138 d), and extensive neuronal pathology was observed including a prominent accumulation of cytoplasmic storage material within the lysosomal-endosomal compartment, a loss of cerebellar Purkinje cells, and widespread axonal degeneration. The CLN2-targeted mouse therefore recapitulates much of the pathology and clinical features of cLINCL and represents an animal model that should provide clues to the normal cellular function of TPP I and the pathogenic processes that underlie neuronal death in its absence. In addition, the CLN2-targeted mouse also represents a valuable model for the evaluation of different therapeutic strategies.

Intracranial Delivery of CLN2 Reduces Brain Pathology in a Mouse Model of Classical Late Infantile Neuronal Ceroid Lipofuscinosis

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a lysosomal storage disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase I (TPP1). Lack of TPP1 results in accumulation of autofluorescent storage material and curvilinear bodies in cells throughout the CNS, leading to progressive neurodegeneration and death typically in childhood. In this study, we injected adeno-associated virus (AAV) vectors containing the human CLN2 cDNA into the brains of CLN2−/− mice to determine therapeutic efficacy. AAV2CUhCLN2 or AAV5CUhCLN2 were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age, and mice were then killed at 13 weeks after injection. Mice treated with AAV2CUhCLN2 and AAV5CUhCLN2 contained TPP1 activity at each injection tract that was equivalent to 0.5- and 2-fold that of CLN2+/+ control mice, respectively. Lysosome-associated membrane protein 1 immunostaining and confocal microscopy showed intracellular targeting of TPP1 to the lysosomal compartment. Compared with control animals, there was a marked reduction of autofluorescent storage in the AAV2CUhCLN2 and AAV5CUhCLN2 injected brain regions, as well as adjacent regions, including the striatum and hippocampus. Analysis by electron microscopy confirmed a significant decrease in pathological curvilinear bodies in cells. This study demonstrates that AAV-mediated TPP1 enzyme replacement corrects the hallmark cellular pathologies of cLINCL in the mouse model and raises the possibility of using AAV gene therapy to treat cLINCL patients.

Membrane topology of CLN3, the protein underlying Batten disease

Juvenile neuronal ceroid lipofuscinosis, or Batten disease, is an autosomal recessive disorder characterized by progressive loss of motor and cognitive functions, loss of vision, progressively severe seizures, and death. The disease is associated with mutations in the gene CLN3, which encodes a novel 438 amino acid protein, the function of which is currently unknown. Protein secondary structure prediction programs suggest that the CLN3 protein has five to seven membrane‐spanning domains (MSDs). To distinguish among a number of hypothetical models for the membrane topology of CLN3 we used in vitro translation of native, Flag epitope‐labeled and glycosylation site‐mutated CLN3 protein in the presence or absence of canine pancreatic microsomes. These were immunoprecipitated using antibodies specific for Flag or peptide sequences within CLN3 or left untreated. The results indicate that CLN3 contains five MSDs, an extracellular/intraluminal amino‐terminus, and a cytoplasmic carboxy‐terminus.

Inclusions in frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP) and amyotrophic lateral sclerosis (ALS), but not FTLD with FUS proteinopathy (FTLD-FUS), have properties of amyloid

TDP-43 and FUS normal cytoplasmic functions are thought to involve regulated aggregation and disaggregation [1, 5, 8], similar to that of prion proteins, where aggregation occurs by a self-templating process, likely involving properties of amyloid, or beta pleated sheet structure. Both thioflavin-T and thioflavin-S fluoresce when bound to amyloid fibrils [7]. We previously showed that TDP-43 peptides form amyloidogenic fibrils, binding to thioflavin-T [3]. Recently, TDP-43-positive lower motor neuron (LMN) inclusions in 28% of 47 cases of ALS, but no inclusions in 22 FTLD-TDP cases, were shown to be positive with thioflavin-S [6]. Using thioflavin-S, we surveyed brain tissues from 44 cases, including FTLD-TDP type A, (17 cases), type B (14 cases), type C (3 cases), sporadic ALS (2 cases) familial ALS (FALS) (2 each with SOD1 and C9orf72 mutations), and FTLD-FUS, including atypical FTLD-U (aFTLD-U, 2 cases) and basophilic inclusion body disease (BIBD, 2 cases). Our routine modified thioflavin-S stain includes pretreatment of tissue sections with potassium permanganate and bleaching with potassium metabisulfite and oxalic acid followed by treatment with sodium hydroxide and hydrogen peroxide, removing lipid autofluorescence and resulting in improved definition of pathological lesions. Slides were viewed using a Nikon BV-2A filter cube. Confocal microscopy was performed using thioflavin-S staining as above and phospho TDP43 (pS409/410-1). Inclusions in most cases of FTLD-TDP and ALS were thioflavin-S positive. The density and distribution of thioflavin-S positive inclusions was similar to that seen with ubiquitin and fewer than with TDP-43 immunohistochemistry. In FTLD-TDP type A, neuronal intranuclear inclusions (NIIs), neuronal cytoplasmic inclusions (NCIs) and dystrophic neurites (DNs) were strongly fluorescent (Fig. 1a). Fluorescent confocal microscopy showed co-localization of thioflavin-S with TDP-43 positive inclusions (Fig. 1b). In FTLD-TDP type B, rare NCIs in all layers of cortex were positive but dentate gyrus inclusions were easily seen (Fig. 1c). In FTLD-TDP type C, long DNs in cortex were strongly thioflavin-S positive (Fig. 1d). Inclusions in FTLD-FUS were negative with thioflavin-S; while rare hippocampal dentate gyrus neurons had fluorescent granular inclusions, large cytoplasmic inclusions and intranuclear vermiform inclusions were not seen with thioflavin-S (Fig. 2). Skein-like inclusions (SLI) and Lewy-like bodies (LLB) in LMNs of ALS cases were positive with thioflavin-S (Fig. 3). The two cases of FALS with SOD1 mutations were not thioflavin-S positive. Additionally, eight of the 17 cases of FTLD-TDP type A had exuberant thioflavin-S positive astrocytosis, making identification of TDP-43 pathology problematic in five of these (Fig. 4). Fig. 1 Superficial frontal cortex, FTLD-TDP type A (1a). Numerous thioflavin-S positive c-shaped and annular NCIs and a few DNs. Inset: thioflavin-S positive NII. Thioflavin-S, 400x. Confocal microscopy (1b). Same case as Fig. 1a, with thioflavin-S (green, left), ... Fig. 2 Motor cortex, FTLD-FUS (BIBD) (2a). Large cytoplasmic upper motor neuron inclusion on left, consistent with basophilic inclusion body (BIB), negative with thioflavin-S. BIB on right immunolabeled with FUS for comparison. Thioflavin-S stain and FUS immunohistochemistry, ... Fig. 3 Lumbar anterior horn, ALS. Lower motor neurons with LLB (left) and SLIs (middle and right). Scattered shrunken neurons nearby (middle and right images) contain compressed SLIs. Thioflavin-S, 600x. Fig. 4 Superficial frontal cortex, FTLD-TDP type A. Exuberant thioflavin-S positive astrocytosis. Thioflavin-S, 200x. There are at least three possible reasons that these results differ from those of Robinson et al. The modified thioflavin-S protocol used in the current study provides results that are superior to other thioflavin-S protocols, the BV-2A filter produces the brightest images, and the brain tissue is briefly paraformaldehyde-fixed (30 hours). Fixation does not likely impact results, but we cannot exclude that possibility. Clearly more cases need to be analyzed with thioflavin-S, and that work is in progress. Nonetheless, it is important that thus far, inclusions in most cases of FTLD-TDP, but not FTLD-FUS or SOD1 FALS, are thioflavin-S positive, suggesting that the prion-like property of TDP-43 may be involved in FTLD-TDP and sporadic ALS and non-SOD1 FALS, but that this may not be the case in FTLD-FUS. The significance of the thioflavin-S positive astrocytosis in some cases of FTLD-TDP type A is currently under investigation. In addition, the fact that TDP-43 in FTLD-TDP has properties of amyloid has implications for the interpretation of amyloid imaging studies [2], as Pittsburgh Compound-B is a modified thioflavin-T derivative [4].

A Knock-In Reporter Model of Batten Disease

Juvenile neuronal ceroid lipofuscinosis is a severe inherited neurodegenerative disease resulting from mutations in CLN3 (ceroid-lipofuscinosis, neuronal 3, juvenile). CLN3 function, and where and when it is expressed during development, is not known. In this study, we generated a knock-in reporter mouse to elucidate CLN3 expression during embryogenesis and after birth and to correlate expression and behavior in a CLN3-deficient mouse. In embryonic brain, expression appeared in the cortical plate. In postnatal brain, expression was prominent in the cortex, subiculum, parasubiculum, granule neurons of the dentate gyrus, and some brainstem nuclei. In adult brain, reporter gene expression waned in most areas but remained in vascular endothelia and the dentate gyrus. Mice homozygous for Cln3 deletion showed two hallmark pathological features of the neuronal ceroid lipofuscinosises: autofluorescent inclusions and lysosomal enzyme elevation. Moreover, CLN3-deficient reporter mice displayed progressive neurological deficits, including impaired motor function, decreased overall activity, acquisition of resting tremors, and increased susceptibility to pentilentetrazole-induced seizures. Notably, seizure induction in heterozygous mice was accompanied by enhanced reporter expression. This model provides us with the unique ability to correlate expression with pathology and behavior, thus facilitating the elucidation of CLN3 function and the pathogenesis of Batten disease.

Frontotemporal lobar degeneration with TDP‐43 proteinopathy and chromosome 9p repeat expansion in C9ORF72: clinicopathologic correlation

Mutations in C9ORF72 resulting in expanded hexanucleotide repeats were recently reported to be the underlying genetic abnormality in chromosome 9p‐linked frontotemporal lobar degeneration with TAR DNA‐binding protein of 43 kD (TDP‐43) proteinopathy (FTLD‐TDP), amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration with motor neuron disease (FTLD‐MND). Several subsequent publications described the neuropathology as being similar to that of FTLD‐TDP and ALS without C9ORF72 mutations, except that cases with mutations have p62 and ubiquitin positive, TDP‐43 negative inclusions in cerebellum, hippocampus, neocortex, and basal ganglia. The identity of this protein is as yet unknown, and its significance is unclear. With the goal of potentially uncovering the significance of these inclusions, we compared the clinical, pathologic and genetic characteristics in cases with C9ORF72 mutations to those without. We confirmed the apparent specificity of p62 positive, TDP‐43 negative inclusions to cases with C9ORF72 mutations. In hippocampus, these inclusions correlated with hippocampal atrophy. No additional correlations were uncovered. However, this is the first report to show that although most cases with C9ORF72 mutations were TDP type B, some of the pathologic characteristics in these cases were more similar to TDP types A and C than to type B cases. These include greater cortical and hippocampal atrophy, greater ventricular dilatation, more neuronal loss and gliosis in temporal lobe and striatum, and TDP‐43 positive fine neuritic profiles in the hippocampus, implying that the C9ORF72 mutation modifies the pathologic phenotype of FTLD‐TDP type B.